Proper genetic and epigenetic regulation of the sex chromosomes during spermatogenesis is crucial for the development of normal male fertility. The sex chromosomes also play a central role in the evolution of hybrid male sterility between species, but the developmental causes of these incipient reproductive barriers remain unclear. The proposed research will use quantitative genetic and genomic experiments in house mice to bridge significant gaps in our understanding of the developmental underpinnings of male sterility. Mice are the predominant genetic models for human reproductive biology, thus male sterility can be studied in greater detail in mice than in most other systems. Here we focus on two closely related mouse species that are partially isolated by hybrid male sterility, providing an ideal model system for studying the consequences of natural genetic divergence on the progression of spermatogenesis. One of our central goals is to test the long- standing hypothesis that regulatory disruption of X-inactivation during spermatogenesis plays a central role in the evolution of hybrid sterility. Towards this end, we are proposing four synergistic research projects. First, we will use cutting-edge sequencing approaches to generate complete genomic sequences for our study organisms. Second, we will use powerful methods of targeted cellular enrichment to study gene expression across key stages of spermatogenesis in two species of mice and their sterile hybrid males. These data will be used to determine if the disruption of X-linked gene regulation during the later stages of spermatogenesis is a primary developmental cause of hybrid male sterility in mice. We will also use these data to identify candidate genes involved in the underlying genetic interactions that disrupt spermatogenesis. Third, we will use additional genetic experiments to directly test if candidate incompatibilities do indeed interact with the X chromosome to cause sterility. Fourth, we will use quantitative genetic methods to further dissect one set of incompatibilities where one or more of the interacting genes that cause hybrid male sterility remain polymorphic within one of the species. This final set of experiments will utilize the extensive genetic resources of the mouse system to study the evolution of hybrid incompatibilities at their earliest possible stage - prior to their fixation between species. Collectively, these experiments will provide important insights into the developmental causes of male sterility.